Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak (Tectona grandis L.f.)

Abstract

BackgroundTeak (Tectona grandis L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world. Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive technique employed for quantifying gene expression levels. One or more appropriate reference genes are crucial to accurately compare mRNA transcripts through different tissues/organs and experimental conditions. Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak. To date, qRT-PCR reference genes have not been identified and validated for teak.ResultsIdentification and cloning of nine commonly used qRT-PCR reference genes from teak, including ribosomal protein 60s (rp60s), clathrin adaptor complexes medium subunit family (Cac), actin (Act), histone 3 (His3), sand family (Sand), β-Tubulin (Β-Tub), ubiquitin (Ubq), elongation factor 1-α (Ef-1α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak. Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene. Their stability as reference genes was validated by NormFinder, BestKeeper, geNorm and Delta Ct programs. Results obtained from all programs showed that TgUbq and TgEf-1α are the most stable genes to use as qRT-PCR reference genes and TgAct is the most unstable gene in teak. The relative expression of the teak cinnamyl alcohol dehydrogenase (TgCAD) gene in lignified tissues at different ages was assessed by qRT-PCR, using TgUbq and TgEf-1α as internal controls. These analyses exposed a consistent expression pattern with both reference genes.ConclusionThis study proposes a first broad collection of teak tissue and organ mRNA expression data for nine selected candidate qRT-PCR reference genes. NormFinder, Bestkeeper, geNorm and Delta Ct analyses suggested that TgUbq and TgEf-1α have the highest expression stability and provided similar results when evaluating TgCAD gene expression, while the commonly used Act should be avoided.

Characterization of Cinnamyl Alcohol Dehydrogenase gene family in lignifying tissues of Tectona grandis L.f.

“…By quantitative real-time PCR, different expression levels of all TgCAD genes were detected for all tissues sampled (Figure 4). Ef1α was the endogenous control for this experiment (Galeano et al 2014). TgCAD1 and TgCAD3 were expressed at very low level in tissues, and TgCAD1 showed lower expression level in leaf and stem secondary xylem at both ages.…”

“…The StepOnePlus™ System (Applied Biosystems, USA) was used for the PCR reactions as follows: 2 min at 50˚C, 2 min at 95˚C, 45 cycles of 15 s at 95˚C, 1 min at 65˚C, using 96-well optical reaction plates (Applied Biosystems, USA). Each reaction was done with technical replicates, with negative control (absence of template); leaf was used as calibrator and EF1α as control gene (Galeano et al 2014). Statistical analyses were performed in SAS at 95 % confidence level using F-test for ANOVA and LSD for pair comparison.…”

“…RNA quality was assessed performing an agarose gel and by Nanodrop ND-1000 Spectrophotometer (NanoDrop Technologies Inc., USA). Also, a PCR reaction was carried out to ensure absence of DNA contamination, using the primers and conditions described by Galeano et al (Galeano et al 2014) to amplify TgEF1α gene. PCR products were analyzed by electrophoresis on a 1 % (w/v) agarose gel with ethidium bromide staining, expecting absence of bands.…”

“…Natural populations of teak have been found in Laos, Myanmar, Thailand, Java Islands and India, with a worldwide planted area and natural forest of 33,381 ha (2.5 million m 3 of wood) (Kollert and Cherubini 2012). Unfortunately, despite its importance, there is a lack of genetic studies regarding gene expression and characterization (Alcântara and Veasey 2013;Galeano et al 2014;Galeano et al 2015). Consequently, to obtain CAD members related to lignin biosynthesis in teak, this study amplified, cloned and characterized three partial CAD genes (TgCAD2, TgCAD3 and TgCAD4) and a complete bona fide CAD gene (TgCAD1).…”

Efficient and new method for Tectona grandis in vitro regeneration

“…Teak grows naturally across the Indian subcontinent through Myanmar and Thailand to Laos, and it is common in deciduous forests and well-drained alluvial soils (Pandey and Brown 2000). Central characteristics of its wood include resistance to weathering, strong fiber, and special oil content, which produces premium timber (Feroz et al 2013), making teak one of the world's most valuable hardwood species (Galeano et al 2014). It is cultivated in more than 36 countries with 5.7 million hectares planted worldwide (Santos et al 2014).…”